The growth of agricultural crops is almost always limited by the availability of nitrogen, and at least 50% of global needs are met by the application of synthetic fertilisers in the form of ammonia, nitrate or urea. Apart from recycling of crop residues and animal manure, and atmospheric deposition, the other most important source of nitrogen for agriculture comes from biological nitrogen fixation.
A small percentage of prokaryots, the diazotrophs, produce nitrogenases and are capable of nitrogen fixation. Members of this group, belonging to the Rhizobiaceae family (for example Mesorhizobium loti, Rhizobium meliloti, Bradyrhizobium japonicum, Rhizobium leguminosarum by viceae) here collectively called Rhizobium or Rhizobia spp and the actinobacterium Frankia spp, can form endosymbiotic associations with plants conferring the ability to fix nitrogen. Although many plants can associate with nitrogen fixing bacteria, only a few plants, all members of the Rosid I Clade, form endosymbiotic associations with Rhizobia spp and Frankia spp., which are unique in that most of the nitrogen is transferred to and assimilated by the host plant. Legumes, including soybean, bean, pea, peanut, chickpea, cowpea, lentil, pigeonpea, alfalfa and clover, are the most agronomically important members of this small group of nitrogen-fixing plants. The rhizobial-legume interaction is generally host-strain specific, whereby successful symbiotic associations only occur between specific rhizobial strains and a limited number of legume species. The specificity of this interaction is determined by chemical signalling between plant and bacteria, which accompanies the initial interaction and the establishment of the symbiotic association (Hirsch et al. 2001, Plant Physiol. 127: 1484-1492). Specific (iso)flavanoids, secreted into the soil by legume spp, allow Rhizobium spp to distinguish compatible hosts in their proximity and to migrate and associate with roots of the host. In a compatible interaction, the (iso)flavanoid perceived by the Rhizobium spp, interacts with the rhizobial nodD gene product, which in turn leads to the induction of rhizobial Nod-factor synthesis. Nod-factor molecules are lipo-chitin-oligosaccharides, commonly comprising four or five β-1-4 linked N-acetylglucosamines, with a 16 to 18 carbon chain fatty acid n-acetylated on the terminal non-reducing sugar. Nod factors are synthesised in a number of variants, characterised by their chemically different substitutions on the chitin backbone which are distinguished by the compatible host plant. The perception of Nod-factors by the host induces invasion zone root hairs, in the proximity of rhizobial cells, to curl and entrap the bacteria. The adjacent region of the root hair plasma membrane invaginates and new cell wall material is synthesized to form an infection thread or tube, which serves to transport the symbiotic bacteria through the epidermis to the cortical cells of the root. Here the cortical cells are induced to divide to form a primordium, from which a root nodule subsequently develops. In legumes belonging to genera like Arachis (peanut), Stylosantos and Sesbania, infection is initiated by a simple “crack entry” through spaces or cavities between epidermal cells and lateral roots. In spite of these differences, perception of Nod factors by the host plant simultaneously induces the expression of a series of plant nodulin genes, which control the development and function of root nodules, wherein the rhizobial endosymbiotic association and nitrogen fixation are localised. A variety of molecular approaches have identified a series of plant nodulin genes which play a role in rhizobial-legume symbiosis, and whose expression is induced at early or later stages of rhizobial infection and nodule development (Geurts and Bisseling, 2002, Plant Cell supplement S239-249). Furthermore, plant mutant studies have revealed that a signalling pathway must be involved in amplifying and transducing the signal resulting from nod-factor perception, which is required for the induction of nodulin gene expression. Among the first physiological events identified in this signal transduction pathway, which occurs circa 1 min after Nod-factor application to the root epidermis, is a rapid calcium influx followed by chloride efflux, causing depolarisation of the plasma membrane and alkalization of the external root hair space of the invasion zone. A subsequent efflux of potassium ions allows re-polarisation of the membrane, and later a series of calcium oscillations are seen to propagate the signal through the root hair cell. Pharmacological studies with specific drugs, which mimic or block Nod-factor induced responses, have identified potential components of the signalling pathway. Thus mastoparan, a peptide which is thought to mimic the activated intracellular domain of G-protein coupled receptors, can induce early Nod gene expression and root hair curling. This suggests that trimeric G protein may be involved in the Nod-factor signal transduction pathway. Analysis of a group of nodulation mutants, including some that fail to show calcium oscillations in response to Nod-factor signals, has revealed that in addition to the lack of nodulation, these mutants are unable to form endosymbioses with arbuscular mycorrhizal fungi. This implies that a common symbiotic signal transduction pathway is shared by two types of endosymbiotic relationships, namely root nodule symbiosis, which is largely restricted to the legume family, and arbuscular mycorrhizal symbiosis, which is common to the majority of land plant species. This suggests that there may be a few key genes which dispose legumes to engage in nodulation, and which are missing from crop plants such as cereals.
The identification of these key genes, which encode functions which are indispensable for establishing a nitrogen fixing system in legumes, and their transfer and expression in non-nodulating plants, has long been a goal of molecular plant breeders. This could have a significant agronomic impact on the cultivation of cereals such as rice, where production of two harvests a year may require fertilisation with up to 400 kg nitrogen per hectare. In accordance with this goal, WO02102841 describes the gene encoding the NORK polypeptide, isolated from the nodulating legume Medicago sativa, and the transformation of this gene into plants incapable of nitrogen fixation. The NORK polypeptide and its homologue/orthologue SYMRK from Lotus japonicus (Stracke et al 2002 Nature 417:959-962), are transmembrane receptor-like kinases with an extracellular domain comprising leucine-rich repeats, and an intracellular protein kinase domain. Lotus japonicus mutants, with a non-functional SYMRK gene, fail to form symbiotic relationships with either nodulating rhizobia or arbuscular mycorrhiza. This implies that a common symbiotic signalling pathway mediates these two symbiotic relationships, where SYMRK comprises an early step in the pathway. The symRK mutants retain an initial response to rhizobial infection, whereby the root hairs in the susceptable invasion zone undergo swelling of the root hair tip and branching, but fail to curl. This suggests that the SYMRK protein is required for an early step in the common symbiotic signalling pathway, located downstream of the perception and binding of microbial signal molecules (e.g. Nod-factors), that leads to the activation of nodulin gene expression.
The search for key symbiosis genes has also focussed on ‘candidate genes’ encoding receptor proteins with the potential for perceiving and binding Nod-factors or surface structures on rhizobial bacteria. U.S. Pat. No. 6,465,716 discloses NBP46, a Nod-factor binding lectin isolated from Dolichos biflorus roots, and its transgenic expression in transformed plants. Transgenic expression of NBP46 in plants is reported to confer the ability to bind to specific carbohydrates in the rhizobial cell wall and thereby to bind these bacteria and utilise atmospheric nitrogen, as well as conferring apyrase activity. An alternative approach to search for key symbiosis genes has been to screen for Nod-factor binding proteins in protein extracts of plant roots. NFBS1 and NFBS2 were isolated from Medicago trunculata and shown to bind Nod-factors in nanomolar concentrations, however, they both failed to exhibit the Nod-factor specificity characteristic of rhizobial-legume interactions (Geurts and Bisseling, 2002 supra).
The Nod-factor binding element, which is responsible for strain specific Nod-factor perception is not, as yet, identified. The isolation and characterisation of this element and its respective gene(s) would open the way to introducing Nod-factor recognition into non-nodulating plants and thereby the potential to establish Rhizobium-based nitrogen fixation in important crop plants.
Rhizobial strains produce strain-specific Nod-factors, lipochitin oligosaccharides (LCOs), which are required for a host-specific interaction with their respective legume hosts. Lotus and peas belong to two different cross-inoculation groups, where Lotus develops nodules after infection with Mesorhizobium loti, while pea develops nodules with Rhizobium leguminosarum by viceae. Cultivars belonging to a given Lotus sp also vary in their ability to interact and form nodules with a given rhizobial strain. Perception of Nod-factor secreted by Rhizobium spp bacteria, as the first step in nodulation, commonly leads to the initiation of tens or even hundreds of rhizobial infection sites in a root. However, the majority of these infections abort and only in a few cases do the rhizobia infect the nodule primordium. The frequency and efficiency of the Rhizobium-legume interaction leading to infection is known to be influenced by variations in Nod-factor structure. The genetics of Nod-factor synthesis and modification of their chemical structure in Rhizobium spp have been extensively characterised. An understanding of Nod-factor binding and perception, and the structure of its component elements is needed in order to optimise the host Nod-factor response. This information would, in turn, provide the necessary tools to breed for enhanced efficiency of nodulation and nitrogen fixation in current nitrogen-fixing crops.
The importance of this goal is clearly illustrated by the performance of the major US legume crop, soybean, which is grown on 15%, or more, of agricultural land in the US. While nitrogen fixation by soybean root nodules can assimilate as much as 100 kg nitrogen per hectare per year, these high levels of nitrogen assimilation are insufficient to support the growth of the highest yielding modern soybean cultivars, which still require the application of fertiliser.
In summary, there is a need to increase the efficiency of nodulation and nitrogen fixation in current legume crops as well as to transfer this ability to non-nodulating crops in order to meet the nutritional needs of a growing global population, while minimising the future use of nitrogen fertilisers and their associated negative environmental impact.